Ministry of Education Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.

Abstract

The perception of lipopolysaccharides (LPS) by plant cells can lead to nitric oxide (NO) production and defense gene induction. However, the signaling cascades underlying these cellular responses have not yet been resolved. This work investigated the biosynthetic origin of NO and the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) to gain insight into the mechanism involved in LPS-induced resistance of Arabidopsis (Arabidopsis thaliana). Analysis of inhibitors and mutants showed that LPS-induced NO synthesis was mainly mediated by an arginine-utilizing source of NO generation. Furthermore, LPS-induced NO caused transcript accumulation of alternative oxidase genes and increased antioxidant enzyme activity, which enhanced antioxidant capacity and modulated redox state. We also analyzed the subcellular localization of NPR1 to identify the mechanism for protein-modulated plant innate immunity triggered by LPS. LPS-activated defense responses, including callose deposition and defense-related gene expression, were found to be regulated through an NPR1-dependent pathway. In summary, a significant NO synthesis induced by LPS contributes to the LPS-induced defense responses by up-regulation of defense genes and modulation of cellular redox state. Moreover, NPR1 plays an important role in LPS-triggered plant innate immunity.

-elicited Arg-utilizing source of generation. A, Effects of mammalian inhibitors and scavenger on level by induction. Protoplasts prepared from wild-type plants were loaded with for 20 min prior to different treatments for 2 h. For each treatment, fluorescence and bright-field images are shown. B, Confocal images of fluorescence in protoplasts from Atnoa1, nia1nia2, cue1, and gsnor1-3 plants treated with control solution or 100 μg mL−1 for 2 h. Bars = 50 µm. C, Quantitative analysis of -related fluorescence by a fluorescence spectrometer under various treatments for 2 h as shown in A and B. WT, Wild type. D, production was examined by analysis. E, Effect of and mammalian inhibitors on -like enzyme activity. F, Effect of on activity of wild-type and nia1nia2 plants. Pr, Protein. Data are means ± se of three experiments. Different letters indicate statistically significant differences between treatments (Duncan’s multiple range test: P < 0.05). [See online article for color version of this figure.]

Induction of PR1 gene expression and callose deposition in Arabidopsis by . A, Approximately 10-d-old transgenic PR1:GUS seedlings grown on medium were then transferred to 24-well plates containing 400 μL of liquid medium without 100 μg mL−1 (control) for 12 h or with 100 μg mL−1 for 6, 12, or 24 h and collected for histochemical GUS staining. Each experiment was performed with eight plants and repeated twice with similar results. B, Quantitative -PCR data showing the expression of the PR1 gene in wild-type (WT) and npr1 Arabidopsis. Total RNA was extracted from the leaves of Arabidopsis after spraying with control solution at 12 h post treatment or 100 μg mL−1 at 0, 6, 12, and 24 h post treatment. Arabidopsis ACTIN2 was used as an internal control. Expression levels for each treatment were normalized to a -treated (6 h) wild-type plant. Values represent means ± se of three independent experiments. C, Callose-staining imaging of leaves and roots from -treated plants. D and E, Callose deposition in leaves (D) and roots (E) was quantified by determining the number of pixels (corresponding to -induced callose) per million pixels in digital photographs. Data are means ± se of three experiments. F, Induction of CalS1 and CalS12 genes in wild-type and npr1 mutant Arabidopsis by treatment. Total RNA was extracted from leaves treated with control solution (−) or 100 μg mL−1 (+) for 12 h. ACTIN2 was used as an internal control. The experiment was performed three times with similar results. G, Quantitative analysis of CalS1 and CalS12 genes shown in F with ImageJ software. Three gel photographs were taken for quantitative analysis, and values represent means ± se. Different letters indicate statistically significant differences between treatments (Duncan’s multiple range test: P < 0.05). [See online article for color version of this figure.]

Model showing the possible signaling pathway for -induced plant innate immunity. The extracellular are recognized by a receptor in the plant cell plasma membrane. perceived by receptors result in an increase in cytosolic Ca2+ (; ), which may activate -like enzyme and then lead to an increase of level. NO functions to potentiate cyclic GMP/Ca2+-dependent PR1 gene expression () as well as up-regulation of antioxidant enzyme activity, and/or promote the nuclear translocation of NPR1 to induce PR1 expression (). perception is mechanistically linked to NPR1-dependent defense responses. Induction by results in the accumulation of and the activation of NPR1. Activated NPR1 then translocates into the nucleus, where it may interact with TGA transcription factors, thus leading to the induction of the PR1 gene, and it also activates the expression of CalS1 and CalS12 genes in the formation of callose by interacting with some unknown transcription factors. These directly induced PR1 expression and callose deposition by constitute plant innate immunity, which protects plants against pathogen infection.